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ORIGINAL PAPER
Gellan gum hybrid hydrogels for the cleaning of paperartworks contaminated with Aspergillus versicolor
Giovanni De Filpo . Anna Maria Palermo .
Riccardo Tolmino . Patrizia Formoso .
Fiore Pasquale Nicoletta
Received: 2 March 2016 / Accepted: 27 July 2016 / Published online: 1 August 2016
� Springer Science+Business Media Dordrecht 2016
Abstract The degradation of archive materials is
related to irreversible phenomena induced by light,
temperature, humidity, air pollution, micro-organ-
isms, and use. Among biological factors, fungi can
induce harmful effects in paper artworks. Further
forms of damage (e.g. artwork swelling, fibre lifting
and sheet delamination) can be caused by water
immersion, which is one of the most commonly used
methods for cleaning paper. To avoid damage it is
necessary to control the amount and absorption rate of
water by paper. Recently, gellan gum hydrogels have
been proposed as effective tools to allow contaminant
removal from paper supports, owing to the controlled
water release and adhesive properties of gellan gum.
In this study hybrid hydrogels were fabricated by
doping gellan gum either with calcium compounds
(calcium sulphate, hydroxide, chloride, and acetate) or
titanium dioxide nanoparticles in order to evaluate
their ability in cleaning different types of paper
samples affected by spots originating from Aspergillus
versicolor. The best decolourization results were
obtained by calcium acetate/gellan gum hydrogels
and titanium dioxide nanoparticle/gellan gum hydro-
gels, while no synergistic effect was found in paper
samples treated with calcium acetate/titanium dioxide/
gellan gum hydrogels. Hybrid hydrogels were tested
on a case-study book.
Keywords Hydrogels � Gellan gum � Aspergillusversicolor � Fungi � Photo-catalysis � Titanium dioxide
Introduction
Paper deterioration
Paper undergoes changes over time, due to modifica-
tions in the molecular structure of its components
(fragmentation and weakening of the polymer chains)
by chemical, physical and biological agents. Ageing is
a natural phenomenon and depends on several factors
including the raw materials (e.g. cellulose, hemicel-
lulose, lignin), the use of additives (e.g. alum, rosin,
dyes, fillers, heavy metals), and manufacture and
storage conditions (delignification, bleaching, pres-
ence of microorganisms, insects, rodents and pollu-
tants, and unsuitable humidity, temperature or light
intensity) (Zervos 2007; Zervos and Alexopoulou
2015).
G. De Filpo (&)Dipartimento di Chimica e Tecnologie Chimiche,
Università della Calabria, 87036 Rende, CS, Italy
e-mail: [email protected]
A. M. Palermo � R. TolminoDipartimento di Biologia, Ecologia, e Scienze della Terra,
Università della Calabria, 87036 Rende, CS, Italy
P. Formoso � F. P. NicolettaDipartimento di Farmacia e Scienze della Salute e della
Nutrizione, Università della Calabria, 87036 Rende, CS,
Italy
123
Cellulose (2016) 23:3265–3279
DOI 10.1007/s10570-016-1021-z
http://crossmark.crossref.org/dialog/?doi=10.1007/s10570-016-1021-z&domain=pdfhttp://crossmark.crossref.org/dialog/?doi=10.1007/s10570-016-1021-z&domain=pdf
Biodeterioration is ‘‘any unwanted change in the
properties of a material caused by the activity of vital
organisms’’ (Hueck 1968). In the case of paper
documents, this change may include both irreversible
transformations of the substrate and aesthetic damage,
which can prevent the correct reading/observation of
the document/artwork. The biodeterioration of paper
is caused by the mechanical and chemical action of
biodeteriogens, e.g. microalgae, bacteria, fungi,
insects.
The mechanical action of some fungi can cause
either micro- or macro-damage, while their metabolic
processes may result in the appearance of very
differently coloured spots/stains, the felting of sup-
ports and an odour change (pungent smell) (Gu-
tarowska et al. 2012). The temperature and relative
humidity affect the metabolism of micro-organisms by
influencing the kinetic rates of chemical and enzy-
matic reactions. High values of temperature and
relative air humidity (20 �C\T\ 45 �C and RHlarger than 60 %) favour the development of fungi.
These microorganisms can cause severe damage to
paper documents as they are able to produce enzymes
capable of hydrolysing a wide variety of natural
polymers, including cellulose (Silva et al. 2006).
The biocidal activity of titanium dioxide
The first research on the photocatalytic destruction of
fungi, specifically the yeast Saccharomyces cerevisiae
Meyen, was carried out by Matsunaga et al. (1985).
The antifungal activity of titanium dioxide has been
examined intensively on other fungal species, namely
Candida albicans (C.P. Robin) Berkhout, Penicillium
expansum Link, Diaporthe niger N.F. Sommer &
Beraha, Aspergillus niger Tiegh, Fusarium solani
(Mart.) Sacc., F. anthophilum (A. Braun) Wollenw, F.
equiseti (Corda) Sacc., F. oxysporum Schltdl., F.
verticillioides (Sacc.) Nirenberg and Penicillium
chrysogenum Thom (Chen et al. 2009; Sichel et al.
2007).
The physical principle of this method is based on
the ability of some semiconductors to accelerate the
radical oxidation of organic substances in the presence
of light (photocatalysis) (De Filpo et al. 2010). The
photocatalytic processes in which a semiconductor
catalyst, e.g. titanium dioxide (TiO2), is involved
when, in contact with water and oxygen, it is irradiated
by photons of suitable energy (i.e. larger than its
bandgap), can be summarized according to the
following reactions:
TiO2 þ hm ! e�cb þ hþvbO2 þ e�cb ! O��2H2Oþ hþvb ! OH� þ Hþ
O��2 þ H2O ! H2O2 ! 2OH�
OH� þ OC ! OCoxOCþ e�cb ! OCred
Here a titanium dioxide molecule absorbs a photon of
energy hm, creating a free electron and electron holepair (ecb
- ? hmb? ) (Hoffmann et al. 1995). Both electron
and hole move to the semiconductor surface and
produce reactive oxygen species, such as O2-� and OH�,
which can oxidize organic compounds (OH� ? -
OC ? OCox), whereas the electrons can reduce them(OC ? ecb
- ? OCred). It is known that some organiccontaminants can be removed if suitably oxidized or
reduced (De Filpo et al. 2015).
Titanium dioxide nanoparticles are considered an
ideal photocatalyst, as they are readily available,
inexpensive, and have a large surface area; however,
they present some difficulties for eventual recovery.
For this reason, titanium dioxide nanoparticles are
generally immobilized on substrates, such as polymer
matrices, glasses, steels, and hydrogels (Nicoletta
et al. 2012).
The TiO2-generated hydroxyl radicals (OH�) and
superoxide anions (O2-�) can inactivate bacteria,
viruses, fungi, and algae by either the photochemical
oxidation of intracellular coenzyme A (Matsunaga
et al. 1988) or damage to the cell membrane (Sunada
et al. 1998) and its polyunsaturated phospholipids
(Gutteridge 1987; Maness et al. 1999).
More recently, it has been found that titanium
dioxide nanoparticles prevent the biodeterioration of
mortars in cultural heritage buildings (Fonseca et al.
2010), inhibit the A. niger colonization of limestone
and Carrara marble (La Russa et al. 2012), and show
interesting antifungal and biocidal properties on wood
(De Filpo et al. 2013) and parchment (De Filpo et al.
2015).
In addition, a cellulosic nanocomposite of TiO2 was
used as a protective and consolidative coating on the
surface of paper fibres. This layered nanocomposite
could protect works of art on paper from the damaging
3266 Cellulose (2016) 23:3265–3279
123
effects of UV light, air pollutants, mould and bacteria.
The reversibility of the consolidation treatment was
tested on blank sheets of paper by washing the
coatings with ethanol (Afsharpour et al. 2011).
Standard methods for cleaning and disinfecting
paper
Fungi play a considerable role in the deterioration of
cultural heritage, as their enzymatic activity induces
fast decay in the raw materials (e.g. paper, leather,
parchment) of historical art objects. In paper conser-
vation, fungi represent an important problem due to
their ability to excrete cellulases, i.e. enzymes that
catalyse the decomposition of cellulose and of some
related polysaccharides into monosaccharides (Ster-
flinger 2010). Consequently, fungal colonization can
result in serious damage to paper documents, such as
weakening, brittleness, discolouration (due to weak
acids produced by fungi) and foxing (due to the
accumulation of reddish and/or brownish stains) (Arai
2000; Michaelsen et al. 2006).
The best way to prevent fungal contamination is to
keep the rooms clean and maintain appropriate
conditions of temperature and humidity (below
55 %) (Valentin 2007; Sterflinger 2010). When con-
tamination is already present, disinfection by physical
or chemical methods is necessary to fight the infection.
Different cleaning methods are available for paper
depending on the particular stains. The mechanical
removal of dust particles and dirt spots (by soft
brushes and cotton pads if they are located on the
support surface, or by scalpels, soft erasers and
brushes with different degrees of hardness if they are
located at depth) is a dry cleaning method, which
avoids possible swelling and bending due to the
absorption of water.
Laser cleaning technology has some advantages
over conventional cleaning methods, being a contact-
less and chemical-free technique (Friberg et al. 1997).
Stains and spots from foxing are generally removed
by washing the paper with demineralized water or with
detergent solutions, or by decolourization using solu-
tions of hydrogen peroxide, acetic acid, oxalic acid,
ammonia, potassium perborate, or sodium and cal-
cium hypochlorite, according to the stain type.
Chemical reactions involving the formation of acids
are the most harmful processes of biodeterioration for
paper, as they can seriously damage the structure of
cellulose (Arai 2000). The optimal pH of paper is
around 7.5, while at a pH below five paper deteriorates
in a short time (Tang 1981). In these cases aqueous de-
acidification is carried out by washing the paper with
basic solutions.
All the methods described in the previous para-
graph are wet treatments and are not always suit-
able for paper artworks. Therefore, in recent years,
researchers have looked for new cleaning and disin-
fection methods in order to achieve a more selective
and less invasive recovery of paper artworks and
books.
In addition to the mechanical removal of biodete-
riogens from paper artefacts, the most commonly used
methods for paper disinfection include chemical and
physical methods. As recently reviewed (Sequeira
et al. 2012), chemical methods can be grouped into
treatments using alcohols, alkylating agents, azole
antifungals, essential oils, phenol derivatives, photo-
catalysts, quaternary ammonium compounds, salts,
and esters of acids. Fumigants, such as ethylene oxide,
have been widely used for the inactivation of
microorganisms in cultural heritage preservation
(Mendes et al. 2007). Sterilization of paper with
ethylene oxide is an effective, but not long-lasting
treatment. In fact, subsequent contamination is possi-
ble as ethylene oxide does not remain as a preventive
biocide on materials (Michaelsen et al. 2006). Never-
theless, being an alkylating agent, ethylene oxide has
been banned in many countries for its carcinogenic
potential (NTP 2014).
The vapours of essential oils can exhibit antifungal
activity against the moulds commonly found on
library and archival materials. It has been shown that
the activity of linalool is fungistatic rather than
fungicidal. Linalool vapours do not affect the bright-
ness of paper or the degree of polymerization of
cellulose, but do reduce the pH of paper (Rakotoni-
rainy and Lavédrine 2005).
Physical methods include dehydration, gamma
irradiation, high-frequency current, low-oxygen envi-
ronments, ultraviolet radiation and temperature
extremes. Alternative techniques are based on silver
nanoparticles (Shirakawa et al. 2013). Gamma radia-
tion at doses ranging between 3 and 15 kGy is highly
effective in the decontamination of fungi, without
causing significant damage to supports such as cellu-
lose depolymerization, increased yellowing, or gen-
eral embrittlement (Adamo and Magaudda 2003).
Cellulose (2016) 23:3265–3279 3267
123
Freeze-drying of paper is a conservation method in
which water is frozen and then removed by sublima-
tion, thus killing conidia and stopping the growth of
fungi and bacteria (Florian 2002). Nevertheless, these
techniques can only be considered decontamination
treatments.
More recently, gellan gum rigid hydrogels have
been investigated as cleaning tools for works of art on
paper (Iannuccelli and Sotgiu 2010). The basic
principle of paper-cleaning by gellan gum hydrogels
is the controlled release of water from gel to paper and
the ability of gellan gum to absorb the surface deposits
and contaminants responsible for the acidic degrada-
tion of paper. The cleaning by gellan gum hydrogels is
based on the trapping and removal of the contaminat-
ing organic material (including hyphae and spores)
by the gel’s three-dimensional network. Gellan
gum is a water-soluble and high-molecular-weight
heteropolysaccharide. It is used as a gelling agent in
biomedical, pharmacological and food applications.
Gellan gum hydrogels are able to retain and control-
lably release large amounts of water. They are stable in
a wide pH range and can be prepared with different
degrees of viscosity (soft and rigid gels, according to
use) by a simple change of gellan gum concentration.
In addition, gellan gum hydrogels can be used in the
restoration and conservation of paper and parchment
artefacts. In fact, since 2003 ICRCPAL, the Italian
Institute for the Restoration and Conservation of
Papers and Books in Rome, has developed wet
cleaning treatments for works of art on paper based
on the use of rigid gellan gels. This technique is a valid
alternative to conventional aqueous solutions, which
can cause irreversible changes in the substrates, such
as discolouration, deformation and fragility. In fact,
the gradual and controlled release of water molecules
from the gel to the paper is less invasive than
immersion or buffing methods, and allows the removal
of the degrading substances according to a process of
concentration gradient, without causing any morpho-
logical changes to the paper. In addition, gellan gum
hydrogels are characterized by easy application and an
effective cleaning action, which guarantees the struc-
tural and aesthetic preservation of paper supports with
no residue on the samples during the treatment and no
rinsing after application (Iannuccelli and Sotgiu
2010).
More recently, the use of gellan gum/titanium
dioxide nanoparticle hybrid hydrogels proved an
effective method to both clean and disinfect parch-
ment contaminated by P. chrysogenum and Cladospo-
rium cladosporioides (Fresen.) G.A. de Vries (De
Filpo et al. 2015).
In the present study, the cleaning properties of pure
gellan gum, gellan gum/de-acidifying substances and
gellan gum/titanium dioxide nanoparticle hybrid
hydrogels on paper samples contaminated by Asper-
gillus versicolor (Vuillemin) Tiraboschi were inves-
tigated. It was expected that the cleaning action of
hydrogels combined with either the de-acidifying
properties of calcium compounds or the photo-
catalytic properties of titanium dioxide nanoparticles
could efficiently clean paper samples containing spots
caused by fungal contamination. In addition, photo-
catalytic disinfection and protection against fungal re-
colonization were expected in samples treated with
gellan gum/titanium dioxide nanoparticle hybrid
hydrogels. In order to explore possible synergistic
effects, paper samples were treated with gellan gum
hybrid hydrogels containing both de-acidifying com-
pounds and titanium dioxide nanoparticles. The hybrid
hydrogels that gave the best results were tested on a
case-study book.
Materials and methods
Paper characterization
The following three types of paper were used:
A. MUNKEN PRINT, which is a wood-free What-
man paper from sustainable forests with low ash
content (Munken Print by Artic Paper, Munkedal,
Sweden).
B. RECYCLED PAPER, which is a high quality
recycled paper, certified FSC, with 60 % recycled
fibres and 40 % recycled post-consumer fibres
(Freelife Cento 70x100 LL80 by Cartiere Fedri-
goni, Verona, Italy).
C. ARCOSET EDITION 1.3, which is made with
eco-friendly paper bleached without the use of
chlorine (Arcoset 70x100 LL80 by Cartiere
Fedrigoni, Verona, Italy).
The sample size was 2 9 2.5 cm2. In addition, the
third page of a textbook (printed in 1953 and
deteriorated by foxing from A. versicolor) was taken
3268 Cellulose (2016) 23:3265–3279
123
into account as a case study. A. versicolor (sequence
JN997427, GenBank) was determined as the culprit of
foxing by DNA sequence analysis (Jurjevic et al.
2012).
The pH of paper aqueous extract was determined
according to the procedure reported by ASTMMethod
D-778 and confirmed by a portable pH meter with a
flat-tip probe designed to optimize surface contact
with leather and paper (HI99171, Hanna Instruments,
Inc., Woonsocket, RI, USA). Briefly, 5 g of paper (cut
into small pieces of about 1 cm2) was boiled in 100 ml
of distilled water for 1 h. Then, the dispersion was
cooled to room temperature (T = 20 �C) and the pHwas measured.
The paper composition was characterized by
Herzberg’s reagent (Houck 2009). Fibres treated with
Herzberg’s reagent are differently coloured according
to their nature. In particular, a purple-red colour
indicates the presence of flax and/or bleached hemp,
while a blue colour is due to pulp flocks, and a yellow
colour reveals the presence of pulp, jute, hemp, and
generally lignin-rich fibres.
Fungal inoculation
The biodeteriogen was A. versicolor from the stock
culture collection of the Laboratory of Plant Biosys-
tematics at the University of Calabria. Although there
are many species frequently found on paper, such as A.
niger, Chaetomium globosum Kunze, C. cladospori-
oides and P. chrysogenum, A. versicolorwas chosen as
it was found to have contaminated the pages of the
case-study book.
The fungal species was grown in solid culture
medium (Malt Extract Agar) in a climatic chamber at
25 �C for 7 days. Once the microorganisms haddeveloped, the conidia were collected and stored in a
saline solution of 0.05 % NaCl. The conidial concen-
tration was 11.3 9 104 conidia/ml as evaluated by a
Thoma counting chamber. Paper samples were placed
in Petri dishes with no addition of any nutritional
substance, in order to ensure microbial growth at the
expense of the paper. Inoculation was performed by
micro-deposition of 50 ll drops of conidia solutionover the sample surface. All samples in Petri dishes
were covered by plastic lids and kept at 25 �C in athermostatic cell with a relative humidity of 80 %.
Two weeks after inoculation, optical microscope
observations (LaborLux 12 POL, Leica, Wetzlar,
Germany) showed the presence of both hyphae and
conidiophores in the specimens.
Then, the paper samples were subjected to various
cleaning test procedures as listed in Table 1, observed
by stereo and optical microscopy, and placed in a
climatic chamber with controlled temperature and
humidity (T = 25 �C and RH = 80 %) for a further15 days to allow possible fungal re-growth. The
biostatic/biocidal action was judged based on the
observations after cleaning and incubation in the
climatic chamber for 15 days. Similar tests were
performed under the harsher conditions of T = 30 �Cand RH = 80 %.
The pictures displayed in the following figures rep-
resent illustrative examples of the images taken with
the optical microscope.
Hydrogel preparation
Hydrogels were prepared from pure gellan gum
solutions (Gelrite�, Sigma-Aldrich, Milan, Italy) and
gellan gum solutions doped with either calcium
compounds (calcium sulphate, hydroxide, chloride,
and acetate, Sigma-Aldrich, Milan, Italy) or titanium
dioxide nanoparticles (P25, Evonik, Essen, Germany)
in order to evaluate their ability in cleaning different
types of paper samples containing spots. Hydrogels
were labelled Gel n, with n ranging from 1 to 7 (see
Table 1), and their thickness was about 5 mm.
Specifically, Gel 1 was simply a pure gellan gum
hydrogel (without any added compound), Gels 2–5
were gellan gum hydrogels doped with calcium
compounds (calcium sulphate, hydroxide, chloride,
and acetate, respectively), Gel 6 was a pure gellan gum
hydrogel (Gel 1) doped with titanium dioxide
nanoparticles and Gel 7 was a calcium acetate-loaded
gellan gum (Gel 5) doped with TiO2 nanoparticles.
Obviously, gellan gum hydrogels must be solid
enough to ensure a controlled water release and easy
manipulation, with no residue on the samples during
the treatments. In previous investigations on parch-
ment (De Filpo et al. 2015), the ideal weight concen-
tration of gellan gum to be used in the solutions was
found to be 3 wt.%. Lower concentrations of gellan
gum gave soft hydrogels with consequent manipula-
tion difficulties and residues on samples, while higher
concentrations of gellan gum resulted in too-rigid
hydrogels with poor cleaning properties. The contact
time between samples and hydrogels was no longer
Cellulose (2016) 23:3265–3279 3269
123
than 1 h in the present study, in order to avoid
excessive water release that could damage the paper.
Gel 1 was prepared in order to compare the cleaning
action of pure gellan gum hydrogels with those of
gellan gum/de-acidifying compounds and gellan gum/
titanium dioxide nanoparticle hybrid hydrogels.
Hydrogels doped with different calcium salts were
prepared using the salt solutions reported in the
literature (Placido 2012). Gellan gum/TiO2 nanopar-
ticle hybrid hydrogels were prepared by placing TiO2nanoparticles in their powder form on the upper face of
the pure gellan gum hydrogels before their complete
gelling (16 mg of TiO2 per g of hydrogel, see Fig. 1).
In this way, hybrid hydrogels with a high loading of
nanoparticles were obtained.
Even if the best cleaning results with gellan gum
gels doped with calcium compounds were obtained by
Gel 3, its strong base characteristics (pH 11.4) did not
suggest the use as a deacidificant tool. So, Gel 7 was
prepared by addition of TiO2 to Gel 5, which is
characterized by a slightly alkaline pH (7.4).
All hybrid hydrogels were stable against 10
swelling–deswelling cycles, and the reproducibility
in their preparation was confirmed by the relatively
constant value of their equilibrium swelling ratio.
In tests involving TiO2 nanoparticles, gels were
UV-irradiated for 5 min (k = 380 nm, power = 10mW/cm2, HPK 125, Philips, Amsterdam, Nether-
lands) in order to activate titanium dioxide. Paper
samples, previously inoculated and incubated with A.
versicolor, were covered with activated gels for 1 h.
Then, hydrogels were gently removed and samples
observed under the optical microscope. The gellan
gum hydrogels were almost transparent to radiation in
the Vis and near-UV regions, and the penetration
depth of the activating radiation was estimated to be
about 1 cm.
Table 1 Chemical composition of cleaning systems and their average cleaning action and biocidal/biostatic activity (±SD)
Gel id Concentration of de-acidifying compound TiO2 Cleaning action (%) Biostatic/biocidal activity (%)
Gel 1 0 0 64 0
Gel 2 [CaSO4] = 40 mg L-1 0 56 0
Gel 3 [Ca(OH)2] = 40 mg L-1 0 85 0
Gel 4 [Ca(Cl)2] = 40 mg L-1 0 52 0
Gel 5 [Ca(CH3COO)2] = 400 mg L-1 0 72 0
Gel 6 0 16 mg/g 90 100
Gel 7 [Ca(CH3COO)2] = 400 mg L-1 16 mg/g 93 100
The concentration of gellan gum water solution was 3 wt.%. Gel 1 was simply a pure gellan gum hydrogel (without any added
compound), Gels 2–5 were gellan gum hydrogels doped with calcium compounds (calcium sulphate, hydroxide, chloride, and acetate,
respectively), Gel 6 was a pure gellan gum hydrogel (Gel 1) doped with titanium dioxide nanoparticles and Gel 7 was a calcium
acetate-loaded gellan gum (Gel 5) doped with TiO2 nanoparticles
(a) (b)
(c)
Fig. 1 a Top view of a gellan gum hydrogel (Gel 1); b top viewand c cross section of a gellan gum hydrogel loaded withtitanium dioxide nanoparticles (16 mg of TiO2/g of hydrogel,
Gel 6). The upper side (that rich in TiO2 nanoparticles) of Gel 6
was placed on the contaminated paper samples. Typical
hydrogel dimensions: length 5 cm, width 5 cm, and height
0.5 cm. The bar size is: a 0.03 cm, b 0.03 cm and c 0.1 cm,respectively
3270 Cellulose (2016) 23:3265–3279
123
Evaluation of cleaning action and biostatic/
biocidal activity
The cleaning action and biostatic/biocidal activity
were evaluated using image treatment software
(Motic Images Plus 2.0). By using the Auto
Segment and Auto Calculation commands it was
possible to separate object regions from the back-
ground using a threshold value, in order to obtain
detailed data from the segmented images, such as
the total area and percentage of total area of the
respective objects. All experiments were performed
at least in triplicate. All statistical analyses were
performed with one-way analysis of variance
(ANOVA) using the Bonferroni post-test (Instat
software, version 3.36 GraphPAD Software Inc., San
Diego, CA, USA) to determine significant differ-
ences in the experimental data. P\ 0.05 wasconsidered statistically significant.
Results
The pH of paper depends on the substances used in its
manufacture and the presence of acid groups as a result
of hydrolysis and oxidation reactions. The experimen-
tal results showed that all the investigated types of
paper had similar pH values. Specifically, the pH
values were
pH paper Að Þ ¼ 8:21� 0:01;
pH paper Bð Þ ¼ 8:55� 0:01;
pH paper Cð Þ ¼ 8:08� 0:01;
where the slightly higher value of paper B can be
explained by its origin as recycled paper (Fedrigoni
2012).
The observation of different paper samples, treated
with Herzberg’s reagent, by polarizing optical micro-
scopy allows their fibre content to be identified
(Fig. 2). The results showed the presence of:
1. Yellow fibres from pulp, jute, hemp, raw, or
generally lignin-rich fibres in paper A samples
(Fig. 2a).
2. Purple-red fibres from flax or bleached hemp, blue
fibres from untreated pulp and yellow fibres from
pulp, jute, hemp, raw, or generally lignin-rich
fibres in paper B samples (Fig. 2b).
3. Only blue fibres from untreated/bleached pulp in
paper C samples (Fig. 2c).
4. Purple-red fibres from flax or bleached hemp in
the book case-study (Fig. 2d).
(a) (b)
(c) (d)
Fig. 2 Fibres fromdifferent paper samples,
treated with Herzberg’s
reagent, imaged by
polarizing optical
microscopy. a Wood-freepaper A, b recycled paper B,c eco-friendly paper C, andd book case-study. The barsize is 30 lm
Cellulose (2016) 23:3265–3279 3271
123
Fungal colonization of the paper samples began about
2 weeks after inoculation. In the case of paper A, made
with wood-free paper and characterized by a heavy
and rough texture, fungi spread both on paper surfaces
and edges, where several hyphae and conidiophores
were evident under the microscope. Therefore, this
type of paper presented an excellent substrate for
fungal growth and consequent fast deterioration.
Paper B is made with recycled paper and charac-
terized by the lightest, smoothest and whitest texture,
while paper C is obtained from eco-friendly cellulose,
i.e. bleached without the use of chlorine, and charac-
terized by a rather yellowish, rough, and wavy aspect.
In both cases paper contamination remained localized
around the initial infection points due to their partic-
ular composition (recycled and eco-friendly paper,
respectively).
Cleaning treatments were carried out by placing the
gellan gum hydrogels listed in Table 1 on inoculated
papers A, B and C. In all tests, the gellan gum
hydrogels left no residues in the paper. Only in the
case of hydrogels containing titanium dioxide (Gels 6
and 7) were some aggregated nanoparticles found by
optical microscope observations after treatment, but
they could be easily removed with a soft brush.
Cleaning action by gellan gum hydrogels (Gel 1)
In this test, paper samples infected with A. versicolor
were covered with pure gellan gum hydrogels (Gel 1)
for 1 h. Gel 1 was able to promote the removal of
particulate matter present on the paper supports as
evaluated by optical microscope observations. Never-
theless, samples placed in the climatic chamber for a
further 15 days showed fungal re-growth.
Cleaning and decolouring action by gellan
gum/calcium compound hybrid hydrogels (Gels
2–5)
In this test, paper samples infected with A. versicolor
were covered with gellan gum/de-acidifying com-
pound hybrid hydrogels (Gels 2–5, prepared with
calcium sulphate, hydroxide, chloride, and acetate,
respectively) for 1 h. Gels 2–5 promoted the removal
of particulate matter on the paper supports due to the
cleaning action of the gellan gum gel, and decoloured
the spots differently according to the calcium
compound used, as shown by the pictures in
Fig. 3a2–d2. Figure 3 shows that all the gels caused
an evident decolouration of the spots present at the
beginning of the experiments (Fig. 3a1–d1), with
better results for Gel 5 (Fig. 3d1, d2), containing
calcium acetate. All treatments were unable to prevent
fungal re-growth on the paper samples after a further
15 days in the climatic chamber (Fig. 3a3–d3). The
results show slight differences in the extent of fungal
re-growth depending on the type of paper (A, B and
C), as shown in Fig. 3d1–f1, d2–f2, and d3–f3.
Cleaning, decolouring action and biostatic/
biocidal activity of gellan gum/TiO2 nanoparticle
hybrid hydrogels (Gel 6)
As can be seen in Fig. 4, the paper samples were
cleaned in an excellent manner by Gel 6, and no fungal
re-growth was observed after a further 15 days in the
climatic chamber (Fig. 4).
Cleaning, decolouring action and biostatic/
biocidal activity of gellan gum/calcium acetate/
TiO2 nanoparticle hybrid hydrogels (Gel 7)
Before applying the gels to the cleaning of a book (the
case study), the cleaning tests were repeated using
calcium acetate-loaded gellan gum/TiO2 nanoparticle
composites in order to evaluate the presence of
synergistic effects. After inoculation with A. versi-
color, paper samples were covered with activated Gel
7 for 1 h. Then, the hydrogels were gently removed
and the samples observed under the optical micro-
scope. As can be seen in Fig. 5, the paper samples
were cleaned in an excellent manner by Gel 7, and no
fungal re-growth was observed after a further 15 days
in the climatic chamber.
Cleaning, decolouring action and biostatic/
biocidal activity of gellan gum hydrogel and gellan
gum/calcium acetate/TiO2 nanoparticle hybrid
hydrogel (Gel 1 and Gel 7) on the case-study book
On the basis of the results of the previous tests (see
section ‘‘Cleaning, decolouring action and biostatic/
biocidal activity of gellan gum/calcium acetate/TiO2nanoparticle hybrid hydrogels (Gel 7)’’), the gellan
3272 Cellulose (2016) 23:3265–3279
123
gum/calcium acetate/TiO2 nanoparticle hybrid hydro-
gel (Gel 7) was tested on the third cover page of the
case-study book, which was characterized by evident
foxing. The page of the case-study book was covered
with Gel 1 (pure gellan gum hydrogel blank) and Gel 7
(activated as previously described) for 1 h. Then, the
hydrogels were gently removed and the page was
observed with a Wood’s lamp. As can be seen from
Fig. 6c, the area treated with Gel 7 shows a better
cleaning than the area treated with Gel 1, as almost all
spots (present on the right of Fig. 6a) are removed,
while several spots are still present after treatment
with Gel 1 (bright spots on the left of Fig. 6c). These
spots are also responsible for an apparent higher
fluorescence arising from the left side of Fig. 6c.
These observations were confirmed by the image
treatment analysis described in section ‘‘Evaluation of
cleaning action and biostatic/biocidal activity’’, which
Gel 1 on paper A
Gel 2 on paper A
Gel 3 on paper A
Gel 4 on paper A
Gel 5 on paper A
Gel 5 on paper B
Gel 5 on paper C
(a1) (a2) (a3)
(b1) (b2) (b3)
(c1) (c2) (c3)
(d1) (d2) (d3)
(e1) (e2) (e3)
(f1) (f2) (f3)
(g1) (g2) (g3)
Fig. 3 a–e Cleaning anddecolouring action by pure
gellan gum hydrogel (Gel 1)
and by gellan gum/de-
acidifying compound hybrid
hydrogels (Gels 2–5) on
wood-free paper A. The first
picture of each row (a1–e1)shows the initial
contaminated condition,
while the second picture
(a2–e2) was taken after thetreatment. The third picture
(a3–e3) shows papersamples after a further
15 days in a climatic
chamber. e–g Cleaning anddecolouring action by gellan
gum/calcium acetate hybrid
hydrogels (Gel 5) on wood-
free paper A, recycled paper
B, and eco-friendly paper C.
The first picture of each row
e1–g1 shows the initialcontaminated condition,
while the second picture e2–g2 was taken after thetreatment. The third picture
e3–g3 shows paper samplesafter a further 15 days in a
climatic chamber. The bar
size in a1 is common to allpictures and 0.2 cm long
Cellulose (2016) 23:3265–3279 3273
123
resulted in an average cleaning action of 92 % for the
area treated with Gel 7 and of 70 % for the area treated
with Gel 1. Nevertheless, Fig. 6c shows the presence
of some ink bleeding around the red letters, may be
due to the particular composition of this ink. So, a
detailed investigation should be generally made on the
Gel 6 on paper A
Gel 6 on paper B
Gel 6 on paper C
(a1) (a2) (a3)
(b1) (b2) (b3)
(c1) (c2) (c3)
Fig. 4 a–c Cleaning, decolouring action, and biostatic/biocidalactivity by gellan gum/TiO2 nanoparticle hybrid hydrogels (Gel
6) on wood-free paper A, recycled paper B, and eco-friendly
paper C. The first picture of each row a1–c1 shows the initial
contaminated condition, while the second picture a2–c2 wastaken after the treatment. The third picture a3–c3 shows papersamples after a further 15 days in a climatic chamber. The bar
size in a1 is common to all pictures and 0.2 cm long
Gel 7 on paper A
Gel 7 on paper B
Gel 7 on paper C
(a1) (a2) (a3)
(b1) (b2) (b3)
(c1) (c2) (c3)
Fig. 5 a–c Cleaning,decolouring action, and
biostatic/biocidal activity by
gellan gum/calcium acetate/
TiO2 nanoparticle hybrid
hydrogels (Gel 7) on wood-
free paper A, recycled paper
B, and eco-friendly paper C.
The first picture of each row
a1–c1 shows the initialcontaminated condition,
while the second picture a2–c2 was taken after thetreatment. The third picture
a3–c3 shows paper samplesafter a further 15 days in a
climatic chamber. No
synergistic effect is evident
from the simultaneous
activity of calcium acetate
and titanium dioxide
nanoparticles. The bar size
in a1 is common to allpictures and 0.2 cm long
3274 Cellulose (2016) 23:3265–3279
123
particular ink composition present in real artefacts in
order to find the optimal number and time of gel
applications.
Discussion
Figure 7 shows optical microscope images of a pure
gellan gum hydrogel (Gel 1) after the cleaning of a
paper sample inoculated with A. versicolor. It is
possible to observe on the hydrogel surface the
presence of both hyaline hyphae and spores (Fig. 7a,
b, respectively), confirming that the hydration and
swelling of samples due to the hydrogels decreases the
adhesion of organic contaminants to the paper
substrates.
The gellan gum concentration of 3 wt.% used in the
hydrogels’ preparation allows their easy manipulation,
and no residue of gellan gum was present on the paper
samples when observed under the optical microscope.
All paper samples benefitted from evident cleaning by
the treatment with pure gellan gum hydrogels (Gel 1),
but not from biostatic/biocidal activity, as after
15 days there was evident fungal re-growth.
The addition of de-acidifying compounds to the
hydrogel formulation (Gels 2–5) results in a decolour-
ing action on the paper samples in addition to the
previously reported cleaning action by pure gellan
gum hydrogels. Decolourization is most effective with
gellan gum/calcium acetate hybrid hydrogels (Gel 5,
see Fig. 3d2), and no particular dependence is
observed on the type of paper sample, as shown in
Fig. 3d2, e2, and f2. Nevertheless, none of the calcium
compounds ensured protection against fungal re-
growth.
The addition of TiO2 nanoparticles into the Gel 1
formulation provides effective cleaning action,
decolourization of the paper support and finally a
biocidal (no fungal re-growth) or, at least, biostatic
activity (no spore development) 15 days after the
treatment. Samples were clearly decolourized and
almost all stains disappeared. In fact, the application of
gellan gum/TiO2 nanoparticle composite hydrogels on
all three types of paper cleaned the dark spots caused
by fungal growth, with the best results obtained on
eco-friendly paper samples.
As reported by Iannuccelli and Sotgiu (2010), the
cleaning mechanism of gels during the treatment can
be summarized as follows:
1. Spontaneous spread of water molecules from the
gel to the paper.
2. Solubilisation of degradation by-products of
cellulose.
Fig. 6 Cleaning, decolouring action, and biostatic/biocidalactivity by gellan gum/calcium acetate/TiO2 nanoparticle
hybrid hydrogels (Gel 7) on the case-study book. a initialcontaminated condition under a Wood’s lamp, b during thetreatment, and c after a further 15 days in a climatic chamberunder a Wood’s lamp. A pure gellan gum hydrogel (Gel 1) was
placed on the left as blank. Gel diameter is 5 cm
Cellulose (2016) 23:3265–3279 3275
123
3. Diffusion of concentrated solution of by-products
from the paper to the gel according to the gradient
of concentration.
4. Effective removal of both surface particulate
matter and a part of the substances that are present
within the paper.
So, a partial penetration to the back of the paper and
some migration of by-products to the sides cannot be
excluded as seen for some red inks in Fig. 6.
Obviously, the penetration and migration of by-
products within the paper artefact is dependent from
the application time and percentage of water present in
the gels, on the degree of porosity and wettability of
the paper, and from the preservation state of the
artefact.
The biostatic/biocidal activity of the hydrogels, due
to the photo-catalytic properties of the incorporated
titanium dioxide nanoparticles, was confirmed by the
absence of fungal re-growth after a further 15 days in
the climatic chamber under the harsher conditions of
T = 30 �C and RH = 80 %.Nevertheless, some TiO2 nanoparticles were found
on paper samples after treatment with gellan gum
hydrogels incorporating titanium dioxide. This appar-
ent drawback could undoubtedly prolong the biocidal
activity of the treatment.
As a consequence of the tests carried out on the
three types of paper, it is possible to confirm the
effectiveness of gellan gum hybrid hydrogels in the
cleaning and decolourization of infected papers. In
particular, hydrogels loaded with either calcium
acetate or titanium dioxide nanoparticles gave the
best results.
Similar results were obtained when the different
types of paper were cleaned by gellan gum/calcium
acetate/TiO2 nanoparticle hybrid composites (Gel 7):
spots were cleaned to a greater extent on the eco-
friendly cellulose paper but Gel 7 did not show any
additional cleaning effect compared with Gel 5 and
Gel 6. Evidently, Gel 7 has similar biostatic/biocidal
properties to Gel 6.
For the tests on the deteriorated book (case study),
Gel 1 and Gel 7 (i.e. the pure gellan gum and gellan
gum/calcium acetate/TiO2 hybrid hydrogels) were
applied simultaneously. Pictures taken after UV
irradiation demonstrate good results with no detri-
mental effect on the inks. The cleaning action and
biostatic/biocidal activity shown by the different
tested hydrogels are summarized in Table 1. To test
the activity of titanium dioxide nanoparticles alone,
paper samples were covered for 1 h with activated
TiO2 nanoparticles after inoculation with A. versi-
color. Then, the titanium dioxide powder was gently
removed from the samples using a soft brush and the
samples were observed under the optical microscope.
No difference in spots was evident between contam-
inated and treated samples, possibly due to the absence
of a wet hydrogel environment, which strongly limits
the decolouring action of TiO2 nanoparticles. Never-
theless, the biostatic/biocidal activity of treatment
with TiO2 nanoparticles was demonstrated by keeping
the sample in the climatic chamber for 15 days, after
which time no fungal re-growth was evident. It is well
known that titanium dioxide under UV irradiation can
cause the oxidation and, consequently, possible
degradation of cellulose and hemicellulose. However,
this possible drawback was mostly avoided in our
(a) (b)
Fig. 7 Optical microscope picture of a pure gellan gumhydrogel (Gel 1) surface after cleaning of a paper sample
inoculated with A. versicolor. It is possible to observe on the
hydrogel the presence of: a hyaline hyphae and b spores of A.versicolor. The bar size is 30 lm
3276 Cellulose (2016) 23:3265–3279
123
investigations, as the activation of TiO2 nanoparticles
in Gels 6 and 7 was performed before their placement
on the paper samples. In order to verify whether the
proposed treatments could change the physical–chem-
ical properties of the tested papers, the water activity,
pH, tensile strength and colour values were checked
2 days after the application of the gellan gum hybrid
hydrogels. No significant difference in these proper-
ties was found in the tested papers. In fact, the values
of the above mentioned physical–chemical properties
changed by\5 % from the initial ones. In particular,water activity values changed by an average 3.3 %
(3.0 % for paper A, 3.7 % for paper B and 3.2 for
paper C, respectively), pH values changed by an
average 1.3 % (1.2 % for paper A, 1.8 % for paper B
and 1.0 for paper C, respectively), paper tensile
strength values changed by an average 2.7 % (2.2 %
for paper A, 3.3 % for paper B and 2.6 for paper C,
respectively) and, finally, whiteness values changed
by an average 4.5 % (4.2 % for paper A, 4.8 % for
paper B and 4.6 % for paper C, respectively).
In this work, the ability of gellan gum hydrogels in
cleaning paper, as well as the capacity of de-acidifying
calcium compounds and titanium dioxide in decolour-
ing paper, were investigated. In particular, the results
showed that the treatment with pure gellan gum
hydrogels ensures the cleaning of paper supports due
to the ‘‘gelled water content’’ of these materials, while
gellan gum/calcium compound hybrid hydrogels
provide both cleaning and decolouring action. An
additional biostatic/biocidal activity was found only in
hybrid hydrogels incorporating titanium dioxide
nanoparticles. Although these results were achieved
using a single fungal species, and further work is
needed to test other species both alone and in
combination in order to simulate real environmental
conditions, hybrid gellan gum hydrogels incorporating
titanium dioxide nanoparticles can be considered an
effective tool to improve the conservation, protection
and usability of library and archive materials. The use
of these hybrid hydrogels is easy and inexpensive, and
allows the single-step cleaning and decolouring of
paper artworks contaminated by A. versicolor.
Conclusions
Paper is the raw material for most of the cultural heritage
preserved in libraries and archives. Since it essentially
consists of cellulose fibres, paper can undergo physical,
chemical and biological processes of degradation. In
particular, biological attacks due to fungi can cause
evident aesthetic damage through the appearance of spots.
Several techniques for cleaning paper have been
proposed and, in this study, the application of rigid
gellan gum hydrogels in combination with either de-
acidifying compounds or titanium dioxide nanoparti-
cles was investigated for the cleaning and decolouring
of paper supports infected by A. versicolor. In
particular, the best results in decolouring spots were
obtained with gellan gum hydrogels enriched with
either calcium acetate or titanium dioxide nanoparti-
cles, although only the photo-catalytic activity of
titanium dioxide was able to inhibit the re-growth of A.
versicolor. No synergistic effect was found in gellan
gum hydrogels incorporating both calcium acetate and
titanium dioxide. Experiments performed on a page of
an old book confirmed that hybrid hydrogels are
respectful of the inks used for book printing. Further
work is in progress in order to test other fungal species
and investigate the long-term effects of the proposed
treatments on the chemical and physical properties of
paper before their applications in real artefacts. In fact,
it is important to emphasize that TiO2 acts through the
generation of reactive species like hydroxyl radicals
and superoxide ions, which would most likely also
have detrimental effects on the paper fibres.
Acknowledgments MIUR, the Italian Ministry forUniversity, is acknowledged for financial supports (Grants
PRIN and EX 60 %).
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Gellan gum hybrid hydrogels for the cleaning of paper artworks contaminated with Aspergillus versicolorAbstractIntroductionPaper deteriorationThe biocidal activity of titanium dioxideStandard methods for cleaning and disinfecting paper
Materials and methodsPaper characterizationFungal inoculationHydrogel preparationEvaluation of cleaning action and biostatic/biocidal activity
ResultsCleaning action by gellan gum hydrogels (Gel 1)Cleaning and decolouring action by gellan gum/calcium compound hybrid hydrogels (Gels 2--5)Cleaning, decolouring action and biostatic/biocidal activity of gellan gum/TiO2 nanoparticle hybrid hydrogels (Gel 6)Cleaning, decolouring action and biostatic/biocidal activity of gellan gum/calcium acetate/TiO2 nanoparticle hybrid hydrogels (Gel 7)Cleaning, decolouring action and biostatic/biocidal activity of gellan gum hydrogel and gellan gum/calcium acetate/TiO2 nanoparticle hybrid hydrogel (Gel 1 and Gel 7) on the case-study book
DiscussionConclusionsAcknowledgmentsReferences